In a recent publication, Saul Sharkis, a professor of oncology at the Hopkins medical school, has developed a novel method for isolating mouse hematopoietic stem cells (HSC), or blood-forming stem cells.
This new strategy will be useful in the field of stem cell biology as a way to better isolate the rare stem cell populations resident in each tissue, or compartment.
Stem cells have the remarkable capabilities of self-renewal, meaning they can regenerate themselves forever and also the ability to differentiate, or become more specialized cells.
A stem cell can be understood to be an immature cell, poised to become other cells that make up the tissues of the body. These stem cells are responsible for maintaining their specific organ or tissue throughout the lifespan of the organism.
For example, HSC can produce all the cells constituting the blood system, including red blood cells and immune cells like macrophages.
It has actually been shown that one HSC can repopulate the entire blood system of a mouse. Armed with this knowledge, researchers are working intensely to create cell-based medical therapies that will one day cure diseases.
Stem cells are a unique population of cells, not only because they maintain tissues, but also because they divide very slowly and are sparsely numbered.
It is these properties which make isolating the stem cells very difficult for researchers.
Traditionally, the isolation of stem cells has focused on specific cell-surface markers, usually proteins that help identify a cell as a particular type.
These proteins can be characteristic of a certain stem cell or can help to enrich the number of stem cells isolated.
Isolation is performed using a fluorescence-activated cell sorter (FACS) that separates individual cells based on these characteristic surface proteins.
As rewarding as this method has been, it is not without its associated problems. Most stem cell surface markers are dynamic and so isolating cells based on a fluctuating population is difficult.
With this problem in mind, Sharkis developed a new approach to isolating HSC using centrifugation and a strategy known as homing, where the stem cells are drawn to a location in the living organism.
The crucial step in the protocol involves removing the animal's bone marrow, the location where most of the HSC reside.
The bone marrow is then separated by centrifugation, or high-speed spinning. This separates the cells based on their density.
Taking the cells at the correct density, they then undergo a process known as lineage depletion. This step separates the HSCs from mature cells, such as red blood cells.
After lineage depletion, this population of cells is stained with a fluorescent dye. The stained cells are then injected into immunocompromised mice, which have immune systems that will not attack the injected cells.
From here, the HSCs "home" to their niche, which is located in the bone marrow. The niche is a protective compartment that regulates stem cells.
After two days of growth in the mice, the bone marrows are flushed once again and the HSCs are isolated using FACS, which sorts cells based on certain characteristic fluorescence.
The cells that are positive for the fluorescent dye are isolated and these are the HSCs which can then be used for scientific research.
This method of FACS is different from previous methods of FACS because it stains the nuclei of the cells and tracks them based on the nuclear stain, not a cell surface-marker stain.
Not only will this method decrease the variability associated with previous isolation methods, it also uses the innate system of the donor mouse to culture and expand the cells.
The novel method created by the researchers will prove invaluable as research into hematopoietic stem cell biology continues.
This method may also be applied to stem cells in other compartments, although it has not yet been tested in these other tissues or organs.
With the promise of stem cell-based therapies, it is imperative to create standardized methods, which isolate stem cells based on their quiescent, or non-dividing, state.
If this is done, medical therapies will be one step closer to reality for patient with life-threatening diseases.
